Immune adjuvant and vaccine composition containing poly-beta-hydroxybutyrate nanoparticles

Abstract

The present invention relates to immune adjuvants and vaccine compositions containing poly-beta-hydroxybutyrate nanoparticles. The nanoparticles based on non-cytotoxic FDA-approved substances according to the present invention have no cytotoxicity, have high biosafety, and can compensate for the disadvantages of BHB. Unlike existing commercialized immune adjuvants, the present invention is a novel nano-sized immune adjuvant based on a bio-derived substance, has versatility with antigens having a protein form without being limited to a specific antigen, and has the convenience of being able to provide an effect in a single mixture form. In addition, the present invention functionally increases the activity of antigen presenting cells (APCs), and induces the activity of follicular helper T cells and germinal center B cells to enhance both effective humoral and cellular immunity, thereby becoming a base substance of a next-generation immune adjuvant.

Description

Immune adjuvant and vaccine composition comprising poly-β-hydroxybutyrate nanoparticles

[0001] The present invention relates to poly-β-hydroxybutyrate nanoparticles having high biosafety while simultaneously having the effect of activating immune cells to increase humoral and cellular immunity, and to an immune adjuvant composition and a vaccine composition comprising the same.

[0002] Immune adjuvants are materials that enhance antigenicity and can be used in vaccine development, or they can be used to treat and prevent diseases by enhancing non-specific immune responses to antigens. Immune adjuvants are primarily used in vaccine manufacturing because they help to rapidly and powerfully sustain immune responses to antigens for extended periods of time.

[0003] Additionally, the use of specific immune adjuvants can modulate the immune response, controlling the subclass of antibodies against the antigen. Immune adjuvants can be used to enhance the immune response, particularly in immunologically immature individuals, young children, or the elderly, with the aim of enhancing the induction of mucosal immunity.

[0004] Most immune adjuvants were discovered through numerous trials and errors while studying natural products. The first research report was in 1925 by Ramon of France, who announced that mixing tapioca, a cassava starch used in food, with diphtheria and tetanus toxoids effectively increased the production of antigen-specific antibodies. Later, the immune-enhancing effect of aluminum complexes was reported, and an effective immune adjuvant in the form of an emulsion containing killed mycobacteria as an immunomodulator was developed. This is also known as Freund's complete adjuvant (FCA), and although it is known to be a very effective immunomodulator, its reactogenicity, a measure of vaccine side effects, is very high, making it unsuitable for human vaccines. Therefore, FIA (Freund's incomplete adjuvant), which does not contain mycobacteria, was developed and approved in the UK. It has been reported that endotoxins from Gram-negative bacteria have an immune-enhancing effect, and in 1974, Ellouz et al. confirmed the effect of muramyl dipeptide (MDP) (Ellouz F. et al., Biochem. Biophys. Res. Commun. 59:1317-25, 1974). Subsequently, lecithin and saponin were also reported to have the potential to act as immune-enhancing adjuvants.

[0005] In this regard, β-hydroxybutyrate (BHB), which is known to be derived from the body and to be able to regulate the activity of immune cells, has a very high biosafety as it is a substance derived from the body, but it has the disadvantage of being highly soluble in water and easily excreted from the body, making it difficult for it to remain in the target area for a long time and act as an immune adjuvant. Accordingly, the inventors of the present invention have developed poly-β-hydroxybutyrate (PHB), a polymer form of BHB, and nanoparticles thereof to solve these shortcomings, and to provide an immune adjuvant composition and a vaccine composition using the same.

[0006] An object of the present invention is to provide a method for producing poly-β-hydroxybutyrate nanoparticles.

[0007] Another object of the present invention is to provide poly-β-hydroxybutyrate nanoparticles manufactured by the above method.

[0008] Another object of the present invention is to provide an immune adjuvant containing the poly-β-hydroxybutyrate nanoparticles.

[0009] Another object of the present invention is to provide a method for increasing the production rate of antibodies to an antigen in a subject by administering the above-described immunoadjuvant composition together with an antigen.

[0010] Another object of the present invention is to provide a vaccine composition comprising the poly-β-hydroxybutyrate nanoparticles and an antigen.

[0011] Another object of the present invention is to provide a method for increasing the production rate of antibodies to an antigen in a subject by administering the vaccine composition.

[0012] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0014] Hereinafter, the present invention will be described in more detail.

[0015] The present invention provides a method for producing poly-β-hydroxybutyrate nanoparticles.

[0016] Specifically, the above manufacturing method may include a step of dissolving poly-β-hydroxybutyrate in dichloromethane and then dropping it into a polyvinyl alcohol (PVA) solution while sonicating it. In addition, the method may further include a step of dropping it again into a polyvinyl alcohol (PVA) solution after the sonication to coat and harden it.

[0017] The present invention also provides poly-β-hydroxybutyrate nanoparticles manufactured by the above manufacturing method.

[0018] The poly-β-hydroxybutyrate nanoparticles of the present invention are characterized by having a size of 50 to 1000 nm, and more preferably, by having a uniform spherical shape of 100 to 200 nm.

[0019] Additionally, the surface of the nanoparticle may be further coated with a specific ligand molecule of a specific cell, and the molecule may be, but is not limited to, lactoferrin or mannose.

[0020] The present invention also provides an immune-enhancing composition comprising antigen-loaded poly-β-hydroxybutyrate nanoparticles.

[0021] The present invention also provides an immune adjuvant containing poly-β-hydroxybutyrate nanoparticles.

[0022] In the present invention, the poly-β-hydroxybutyrate (PHB) is a polymer belonging to the polyester series, which is a polymer form of β-hydroxybutyrate (BHB), and is attracting attention as a bio-derived and biodegradable polymer, and is a substance that has already been approved by the FDA as a safety confirmed substance. β-hydroxybutyrate (BHB) is produced in the liver through fatty acid metabolism and ketone body production amino acid metabolism, and is known to be able to regulate immune cell activity. However, it has the disadvantage of being highly soluble in water and easily excreted from the body, so it is characterized by being difficult to control its activity while remaining in the target area for a long time, by producing PHB nanoparticles. To complement this, the inventors of the present invention developed a novel immune adjuvant that has the advantage of being better delivered to the target area as a nanoparticle while simultaneously maintaining the effect of BHB continuously.

[0023] In the present invention, “adjuvant” means a substance that has a positive effect on the action of another substance, such as pharmacologically or immunologically, with the meaning of assistance or support, and is expressed as an immune adjuvant, immune stimulant, etc. in the present invention.

[0024] The poly-β-hydroxybutyrate nanoparticles of the present invention have the effect of enhancing both effective humoral and cellular immunity by increasing the activity of antigen-presenting cells (APCs).

[0025] In the present invention, "antigen-presenting cells (APC)" refer to cells that endocytose protein antigens and then present antigen-derived peptide fragments together with major histocompatibility complex (MHC) molecules to T cells to activate them. Representative antigen-presenting cells include dendritic cells (DCs), B cells, and macrophages, which are the main immune cells responsible for cellular immunity in the body. When antigens are introduced, these cells internalize them and then display them on their surface, thereby acting as antigen-expressing cells that allow other cells of the immune system (such as T cells) to recognize the antigens.

[0026] Among the above antigen-presenting cells, dendritic cells are known to exist primarily in tissues that come into contact with the external environment, such as the skin, nose, lungs, stomach, or intestinal lining. Specifically, the cells in the skin are called Langerhans cells. Dendritic cells can be found in an immature state in the blood, and upon activation, they are known to migrate to lymphoid organs and interact with T and B cells to initiate an immune response. Furthermore, dendritic cells are known to extend projections called dendrites during a specific developmental stage.

[0027] In the present invention, the type of antigen-presenting cell is not particularly limited, and non-limiting examples thereof may include dendritic cells, Langerhans cells, macrophages, monocytes, or B cells. In one embodiment of the present invention, dendritic cells, seed-derived B cells, and follicular helper T cells are targeted.

[0028] The poly-β-hydroxybutyrate nanoparticles of the present invention are characterized by having a size of 50 to 1000 nm, and more preferably, by having a uniform spherical shape of 100 to 200 nm.

[0029] The present invention also provides an immune adjuvant characterized in that the surface of the poly-β-hydroxybutyrate nanoparticles is coated or labeled with a molecule that is a specific ligand for a target cell.

[0030] The molecule may be, but is not limited to, lactoferrin or mannose.

[0031] The present invention also provides a method for increasing the antibody production rate against an antigen by administering the immune adjuvant together with an antigen to a subject.

[0032] In the present invention, the administration may be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

[0033] The present invention also provides an immune-enhancing composition containing poly-β-hydroxybutyrate nanoparticles loaded with an antigen.

[0034] In the present invention, immune enhancement means increasing the immune response in various specific or non-specific ways, and non-specific substances other than antigens can also enhance the immune response.

[0035] The present invention also provides a vaccine composition comprising the poly-β-hydroxybutyrate nanoparticles and an antigen.

[0036] In the present invention, "antigen" is a general term for all substances that enter a living body and cause an immune response, and generally refers to all substances that are recognized as foreign substances in a living body, induce an immune response state, and react with immune cells or antibodies of a sensitized subject. In the present invention, the "antigen" may be used as a general term synonymous with the term "immunogen", and may mean a molecule that includes one or more epitopes that can stimulate the host immune system to cause a secretory, humoral, or cellular immune response specific to the antigen. In the present invention, non-limiting examples of the antigen may be peptides (including polypeptides and proteins), DNA, siRNA, or mRNA. In the present invention, the antigen included in the vaccine composition may be characterized as being selected from proteins, peptides, nucleosides, nucleotides, viruses, antiviral agents, antitumor agents, antibiotics, and anti-inflammatory agents.

[0037] In the present invention, the vaccine composition may be characterized in that it is for the prevention or treatment of a disease caused by at least one virus selected from the group consisting of avian influenza virus, swine influenza virus, and novel influenza virus, and may be characterized in that it is for the prevention or treatment of at least one disease selected from the group consisting of cervical cancer, skin melanoma, prostate cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, head and neck cancer, vulvar cancer, bladder cancer, brain cancer, and glioma.

[0038] In the present invention, the antigen loading amount and loading efficiency of the poly-β-hydroxybutyrate nanoparticles can be expressed by the following formula, and the loading efficiency of the final nanoparticles is characterized by being 50 to 90%, preferably 80 to 90%.

[0039] Loading amount = Loaded antigen amount / PHB nanoparticle amount X 100%

[0040] Loading efficiency = (loading amount) / (total protein) X 100 (%)

[0041] The present invention also provides a method for increasing the antibody production rate against an antigen by administering the vaccine composition to an animal other than a human.

[0042] In the present invention, the administration may be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

[0043] In the present invention, carriers, excipients, and diluents that may be included in an immune adjuvant or vaccine composition containing poly-β-hydroxybutyrate nanoparticles include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition may be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Formulations for non-oral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0044] The dosage of the immune adjuvant containing poly-β-hydroxybutyrate nanoparticles of the present invention may vary depending on the age, sex, weight, etc. of the subject to be administered, and the dosage of the vaccine may also be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, etc.

[0045] In addition, the poly-β-hydroxybutyrate nanoparticles of the present invention can be used as an additive to a pharmaceutical composition including a preventive or therapeutic vaccine used to prevent and treat cancer, particularly skin melanoma cancer, prostate cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, head and neck cancer, vulvar cancer, bladder cancer, brain cancer, and glioma, as well as non-communicable chronic diseases.

[0046] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0047] The poly-β-hydroxybutyrate nanoparticles of the present invention complement the shortcomings of the existing body-derived substance, β-hydroxybutyrate (BHB), and, as a bio-derived substance, possess extremely high biosafety. Simultaneously, they enhance the activity of antigen-presenting cells and activate immune cells, thereby enhancing both humoral and cellular immunity, making them suitable for diverse applications as next-generation immune adjuvants and vaccine compositions.

[0048] Figure 1 is a schematic diagram showing the process for manufacturing PHB nanoparticles (PHB-NPs).

[0049] Figure 2 is a schematic diagram showing the process for manufacturing FITC-labeled lactoferrin (LF)- or mannose (MN)-coated PHB-NPs.

[0050] Figure 3 is a diagram showing the results of dynamic light scattering analysis of PHB nanoparticles (a) and observation using a transmission electron microscope.

[0051] Figure 4 is a diagram showing the results of observing FITC-labeled lactoferrin (LF)- or mannose (MN)-coated PHB-NPs using a confocal microscope.

[0052] Figure 5 is a diagram showing the results of a cytotoxicity test of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0053] Figure 6 is a diagram showing the effect of poly-β-hydroxybutyrate (PHB) nanoparticles on inducing the activation of T follicle helper cells and germinal center B cells.

[0054] Figure 7 is a diagram showing the effect of poly-β-hydroxybutyrate (PHB) nanoparticles on inducing T cell responses through dendritic cell stimulation.

[0055] Figure 8 is a diagram showing an experimental process for confirming the antigen loading amount and efficiency of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0056] Figure 9 is a diagram showing the results of an experiment to confirm whether the antigen is denatured by heat during the manufacturing process of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0057] Figure 10 is a diagram showing the degree of internalization of poly-β-hydroxybutyrate (PHB) nanoparticles into the dendritic cells.

[0058] Figure 11 is a diagram showing the process and results of an animal experiment to confirm the role of poly-β-hydroxybutyrate (PHB) nanoparticles as antigen carriers.

[0059] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0060] In addition, when describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description is omitted.

[0061] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0062] [Manufacturing example]

[0063] Manufacturing Example 1. Manufacturing of poly-β-hydroxybutyrate (PHB) nanoparticles

[0064] Poly-β-hydroxybutyrate (PHB) was dissolved in dichloromethane (DCM), and then sonicated while dropping into a 2 wt.% polyvinyl alcohol (PVA) solution to produce nanoparticles. Subsequently, the solution was dropped into a 1 wt.% polyvinyl alcohol (PVA) solution to harden the solution, followed by a coating process. PHB nanoparticles (PHB-NPs) were produced through a mixing process to evaporate the DCM, and an ultracentrifugation process.

[0065] Manufacturing Example 2. Manufacturing of PHB-NPs coated with fluorescein isothiocyanate (FITC)-labeled lactoferrin (LF) and mannose (MN).

[0066] In order to impart cell specificity to the particle surface during the production of PHB-NPs, lactoferrin (LF), a specific ligand for brain cells, and mannose (MN), a specific ligand for dendritic cells, a type of immune cell, were labeled with fluorescein isothiocyanate (FITC), respectively, and PHB-NPs coated with FITC-labeled LF and PHB-NPs coated with FITC-labeled MN were produced using a single emulsion method. LF and MN were used as natural emulsifiers instead of polyvinyl alcohol (PVA) used in the nanoparticle production process. As shown in Fig. 2, the production process involved ultrasonic disruption of PHB dissolved in DCM as an oil phase onto FITC-labeled LF or MN dissolved in distilled water as an aqueous phase. Afterwards, the solution was dropped into a 1 wt.-% LF and MN solution dissolved in distilled water, and a curing process was performed. DCM was evaporated through a mixing process (stirring), and FITC-labeled lactoferrin (LF) and mannose (MN) coated PHB-NPs were finally manufactured through an ultracentrifugation process.

[0067] [Example]

[0068] Example 1. Physicochemical properties of poly-β-hydroxybutyrate (PHB) nanoparticles

[0069] When the size of the poly-β-hydroxybutyrate (PHB) nanoparticles produced through the above Manufacturing Example 1 process was measured by dynamic light scattering (DLS), it was measured to be 166.6 nm, confirming that particles of an appropriate nano size were formed. In addition, when the poly-β-hydroxybutyrate (PHB) nanoparticles were observed through a transmission electron microscope (TEM), it was confirmed that uniform, circular nanoparticles were formed (Fig. 3).

[0070] In addition, the PHB-NPs coated with fluorescein isothiocyanate (FITC)-labeled lactoferrin (LF) and mannose (MN) produced through the manufacturing example 2 process were examined by confocal microscopy, and it was confirmed that FITC-labeled LF and MN, which impart cell specificity to the particle surface, were coated on the surface of the PHB-nanoparticles (Fig. 4).

[0071] Example 2. Confirmation of cytotoxicity of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0072] To determine the cytotoxicity of poly-β-hydroxybutyrate (PHB) nanoparticles, spleen cells isolated from 6-8 week-old BALB / c mice were treated with poly-β-hydroxybutyrate (PHB) nanoparticles at various concentrations, and the cytotoxicity was determined by checking the ratio of dead cells using a flow cytometer after staining with 7-aminoactinomycin D (7-AAD) and Annexin V. As a result of the experiment, as can be seen in Fig. 4, it was confirmed that poly-β-hydroxybutyrate (PHB) nanoparticles did not exhibit toxicity to cells.

[0073] Example 3. Confirmation of the immune response-inducing effect of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0074] To confirm the function of PHB nanoparticles as an immune adjuvant, 6-8 week-old BALB / c mice were vaccinated intranasally with the model antigen ovalbumin (OVA) at 2-week intervals. PHB was administered at 2.5 mg per mouse, OVA at 10 μg, and cholera toxin (CT) at 1 μg as a positive control. Enzyme-linked immunosorbent assay (ELISA) results showed that the group administered PHB nanoparticles as an immune adjuvant showed a significant increase in blood antigen-specific antibody IgG compared to the other groups. ELISA analysis of lung lavage fluid from the mice also showed a significant increase in antigen-specific secretory antibody IgA in the group administered PHB nanoparticles as an adjuvant.

[0075] Example 4. Confirmation of the immune cell activation effect of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0076] To confirm the function of PHB nanoparticles as an immune adjuvant, mice were vaccinated intranasally with a model antigen (OVA) at two-week intervals. Two weeks after the last vaccination, germinal center B cells (CD19) important for antigen-specific antibodies were detected in the spleen and lymph nodes of mice. + GL7 + ) and follicular helper T cells (CD4) that regulate these immune responses + CXCR5 + PD-1 + ) clusters were confirmed. As a result of the flow cytometry analysis, it was confirmed that the proportion and number of germinal center B cell clusters significantly increased in the group administered PHB nanoparticles as an adjuvant. It was also confirmed that the proportion and number of follicular helper T cells clusters significantly increased in the group administered PHB nanoparticles as an adjuvant.

[0077] Example 5. Confirmation of the effect of poly-β-hydroxybutyrate (PHB) nanoparticles on inducing T cell responses through dendritic cell stimulation.

[0078] To confirm the mechanism of action of PHB nanoparticles as an immune adjuvant, mouse bone marrow derived dendritic cells (BMDCs) were treated with PHB nanoparticles and a model antigen (OVA) at various concentrations, and the expression of MHCII and costimulatory factors CD80, CD86, and OX40L was confirmed using flow cytometry. As a result, it was confirmed that the expression of MHCII and costimulatory factors CD80, CD86, and OX40L significantly increased depending on the concentration when PHB nanoparticles were treated.

[0079] Example 6. Confirmation of antigen loading capacity and efficiency of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0080] In order to confirm the antigen loading amount and efficiency during the PHB nanoparticle manufacturing process, ovalbumin (OVA) was loaded as a model antigen using a double emulsion method. As shown in Fig. 8, the process of manufacturing OVA-loaded PHB-NPs was as follows: Pluronic, F-127 (water, W1; stabilizer) mixed with OVA (10 mg) was dropped into DCM (oil, O) in which PHB (50 mg) was dissolved, followed by an ultrasonic grinding process (W1 / O). The PHB nanoparticles (W1 / O) that went through this process were dropped into a PVA solution (W2) to create a final W1 / O / W2 emulsion, and then a homogenization process was performed. Afterwards, the OVA-loaded PHB-NPs were collected through the ultracentrifugation process, and the amount of unloaded OVA in the supernatant (32 ml) was measured using a protein quantification method (BCA assay) to calculate the loading efficiency as follows.

[0081] (BCA value = 53 μg / ml, total supernatant volume: 32 ml, unloaded OVA: 53 X 32 = 1.696 (mg))

[0082] Loading amount: Loaded antigen amount / PHB nanoparticle amount X 100% = 8.304 wt.-%)

[0083] Loading efficiency: (loading amount)) / (total protein) X 100 (%) = 8.304 / 10 X 100 (%) = 83.04 wt.-%)

[0084] In addition, since the ultrasonic grinder used to load OVA during the PHB-NPs manufacturing process generates a lot of heat, the process was carried out on ice at a low temperature to prevent denaturation of the antigen due to heat. However, since denaturation of the loaded antigen cannot be ruled out, circular dichroism was analyzed to confirm the secondary structure (α-helix, β-sheet, random coil) of the antigen in the supernatant after nanoparticle production. The α-helix structure is known to have two negative bands at 208 nm and 222 nm, and a positive maximum at 193 nm. The β-sheet structure is known to have a minimum at 218 nm and a maximum at 195 nm. The results of measuring and analyzing circular dichroism confirmed that there was no change in the secondary structure of the antigen even after the sonication process was performed to load the antigen onto the nanoparticle.

[0085] Example 7. Confirmation of intracellular penetration of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0086] To determine whether PHB-NPs are internalized into cells, mBMDCs were treated with FITC-labeled PHB-NPs for 1 hour. Confocal microscopy confirmed that PHB-NPs were internalized into the cells when mouse bone marrow-derived dendritic cells (BMDCs) treated with FITC-labeled PHB-NPs were examined.

[0087] To confirm that PHB-NPs were internalized rather than coated on the outside of the cells, a single layer of cells was observed using z-stack, and as a result, it was confirmed that the color development of FITC increased toward the center of the cells, confirming that PHB-NPs were internalized inside the cells.

[0088] Example 8. Confirmation of the humoral immune response-inducing effect of poly-β-hydroxybutyrate (PHB) nanoparticles.

[0089] To determine whether PHB nanoparticles can function not only as an immune adjuvant but also as a carrier and release loaded antigens in vivo, ovalbumin (OVA)-loaded PHB NPs were intranasally inoculated into mice at two-week intervals. PHB was administered at 2.5 mg per mouse, OVA at 8 μg to determine loading efficiency, and cholera toxin (CT) at 1 μg was administered as a positive control (Fig. 11).

[0090] ELISA results confirmed that the group vaccinated with OVA-loaded PHB NPs produced higher levels of antigen-specific IgG antibodies in the blood compared to the other groups. Therefore, it was confirmed that PHB NPs can also function as a carrier capable of carrying antigens.

Claims

1. A method for producing poly-β-hydroxybutyrate nanoparticles, comprising the step of dissolving poly-β-hydroxybutyrate in dichloromethane (DCM) and then dropping it into a polyvinyl alcohol (PVA) solution while performing sonication.

2. In paragraph 1, A method for producing poly-β-hydroxybutyrate nanoparticles, characterized in that it further comprises a step of coating and curing by dropping the nanoparticles into a polyvinyl alcohol (PVA) solution after ultrasonic grinding.

3. Poly-β-hydroxybutyrate nanoparticles manufactured according to the manufacturing method of paragraph 1 or 2.

4. In paragraph 3, The above nanoparticles are poly-β-hydroxybutyrate nanoparticles characterized by having a size of 95 to 200 nm.

5. In paragraph 3, Poly-β-hydroxybutyrate nanoparticles characterized in that a specific cell-specific ligand molecule is further coated on the surface of the nanoparticles.

6. In paragraph 5, Poly-β-hydroxybutyrate nanoparticles characterized in that the ligand molecule is lactoferrin or mannose.

7. An immune-enhancing composition comprising poly-β-hydroxybutyrate nanoparticles loaded with an antigen.

8. An immune adjuvant comprising poly-β-hydroxybutyrate nanoparticles.

9. In paragraph 8, An immune adjuvant characterized in that the surface of the nanoparticle is further coated with a specific ligand molecule for the target cell.

10. In paragraph 9, An immune adjuvant characterized in that the above molecule is lactoferrin or mannose.

11. A method for increasing the antibody production rate against an antigen by administering the immune adjuvant of Article 8 together with an antigen to an individual.

12. In paragraph 11, A method characterized in that the above administration is performed by any one of the administration methods selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

13. A vaccine composition comprising poly-β-hydroxybutyrate nanoparticles and an antigen.

14. In paragraph 13, A vaccine composition characterized in that a specific ligand molecule for a target cell is further coated on the surface of the nanoparticle.

15. In paragraph 14, A vaccine composition characterized in that the above molecule is lactoferrin or mannose.

16. In paragraph 13, A vaccine composition characterized in that the antigen is selected from proteins, peptides, nucleosides, nucleotides, viruses, antiviral agents, antitumor agents, antibiotics, and anti-inflammatory agents.

17. In paragraph 13, A vaccine composition characterized in that it is for the prevention or treatment of a disease caused by at least one virus selected from the group consisting of avian influenza virus, swine influenza virus, and novel influenza virus.

18. In paragraph 13, A vaccine composition characterized in that it is for the prevention or treatment of at least one disease selected from the group consisting of cervical cancer, skin melanoma, prostate cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, head and neck cancer, vulvar cancer, bladder cancer, brain cancer, and glioma.

19. A method for increasing the antibody production rate against an antigen by administering the vaccine composition of Article 13 to an individual.

20. In paragraph 19, A method characterized in that the above administration is performed by any one of the administration methods selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

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